HLA-E is a ubiquitously expressed non-classical class Ib molecule. Under homeostatic conditions, HLA-E preferentially presents essentially invariant signal peptides of classical class Ia proteins (HLA-A, -B, and -C) to the innate CD94/NKG2 receptors, thereby regulating NK cell activity. HLA-E-restricted regulatory CD8+ T cells may prevent autoimmunity. Of interest, with infections by certain bacteria and viruses, HLA-E displays a remarkably diverse repertoire of pathogen-derived peptides that are sensed by TCR of CD8+ T cells. We report the first HLA-E-restricted HIV Gag epitope (KL9) identified using in vitro-re-stimulated CD8+ T cells from an elite controller. Functional analysis f HLA-E-restricted KL9-specific T cells revealed both effector (degranulation) and regulatory functions (IL-26 production). This work will begin to dissect this yet undefined host immune immunity, particularly to map conserved epitopes and assess the character of these reactivities within the HLA-E based antigen presentation system in HIV infection. This work is important for HIV vaccine development because the virus may have co-opted this mechanism to limit host immunity. A balance between effector and regulatory immunity in the context of HLA-E might allow partial clearance of HIV, thus providing some levels of protection while avoiding excessive inflammation and pathology, albeit at the cost of viral persistence and chronic infection. Understanding how embedded HLA-E-restricted epitopes affect the induction of HIV immunity would be crucial for vaccine design. The lack of HLA-E polymorphism further implies that some HLA-E-bound epitopes may be potential candidates for a universal HIV vaccine, in contrast with those restricted by the highly polymorphic class Ia loci. Specific Aim 1 will map HLA-E-restricted epitopes in conserved domains across the HIV proteome with CD8+ T cells from elite controllers using an established protocol. Epitopes will be verified by mass spectrometry. Individual HIV epitopes are expected to elicit qualitatively distinct CD8+ responses, since the TCR-binding face of HLA-E is basically monomorphic and thus, the bound peptide strongly influences specificity and functions. Specific Aim 2 will examine the effector and regulatory functional profiles and public TCR usage of HLA-E-restricted CD8+ T cells to the newly mapped specificities. Three T cell cultures will be generated from elite controllers or contingently from healthy donors for each epitope mapped. Selection of public TCRs may indicate that a large segment of the human population is capable of responding and these structures can be exploited by new technologies such as TCR affinity maturation to create new classes of therapeutic agents. Specific Aim 3 will be a correlative study of HLA-E-restricted HIV-specific CD8+ T cells in 30 controllers and 40 progressor patients who are off ART. The ability of these HLA-E epitopes to interact with NKG2 receptors will also be assessed with HLA-E tetramers in 10 HIV seropositive individuals. These studies will provide insights into the role of HLA-E-restricted NK and CD8+ T cell surveillance in HIV infection.